The present invention relates generally to compositions and methods for increasing muscle synthesis and treating disease in vertebrate subjects. More particularly, the invention is directed to immunological compositions and methods for reducing myostatin activity in vertebrate subjects.
Livestock producers have traditionally used breeding programs to select animals that yield maximum amounts of protein with acceptable performance as measured by feed efficiency, reproductive function and general health. Cattle which exhibit increased muscle mass due to both hypertrophy and hyperplasia of muscle cells have been observed in a number of breeds. The incidence of this condition, which is referred to as double-muscling, is most pronounced in Belgian Blue cattle. Muscle mass is increased by approximately 20% with a decrease in bone and fat mass in these animals (Shahin and Berg, Can. J. Anim. Sci. (1985) 65:279-293). Belgian Blue cattle also utilize feed efficiently and give rise to a higher percentage of desirable cuts of meat (Casas et al., J. Anim. Sci. (1997) 75(Supp 1):149). Double-muscling in Belgian Blue cattle is inherited and is believed to be recessive since heterozygotes may be normal or have only a modest increase in muscle mass.
Despite the advantages of this condition, double-muscled cattle often have undesirable traits. For example, because calves are generally 10-38% heavier than normal, dystocias are prevalent, requiring cesarean deliveries. Animals also exhibit abnormal reproduction due to poorly developed reproductive tracts and have other anatomical abnormalities such as macroglossia. Other breeds of cattle, such as the Piemontese from northern Italy, have varying degrees of double-muscling and also display many of these undesirable traits.
The double-muscling characteristic identified in some cattle breeds has now been traced to mutations in the myostatin gene (Grobet et al., Nature Genetics (1997) 17:71-74; Kambadur et al., Genome Research (1997) 7:910-915; McPherron and Lee, Proc. Natl. Acad. Sci. USA (1997) 94:12457-12461). This mutation appears to result mainly in an increase in the number of muscle cells (hyperplasia) rather than an increase in the size of individual muscle fibers (hypertrophy). A condition referred to as muscular hypertrophy has also been identified in the Pietrain breed of pig. This condition is not related to the myostatin gene and has been identified as a mutation in a gene responsible for calcium transport.
McPherron et al., Nature (1997) 387:83-90, have identified a member of the transforming growth factor-xcex2 (TGF-xcex2) superfamily of proteins in mice, referred to as growth/differentiating factor-8 (GDF-8). GDF-8 acts as a negative regulator for skeletal muscle growth and is expressed in developing and adult skeletal muscles. Gene knockout experiments in mice have resulted in homozygous mutants which are 30% larger than wild-type mice. This increase in size is due primarily to an increase in muscle mass with individual muscles from the mutants weighing 2-3 times more than those from wild-type mice (McPherron et al., Nature (1997) 387:83-90). McPherron and Lee, Proc. Natl. Acad. Sci. USA (1997) 94:12457-12461 and Grobet et al., Nature Genetics (1997) 17:71-74 evaluated similar genomic sequences in a number of species, including cattle, and reported that double-muscled cattle had defects in the gene coding for a protein highly homologous to GDF-8. This protein is now called myostatin.
Thus, it appears that myostatin is produced by muscle cells and regulates the proliferation and differentiation of myoblasts. In Belgian Blue and Piemontese cattle, natural defects in the gene are believed to result either in production of an abnormal protein or a reduced amount of myostatin, either of which has the effect of increasing muscle growth.
The myostatin gene from a number of vertebrate species, including mouse, rat, human, baboon, cattle, pig, sheep, chicken, turkey, and zebrafish has been identified and the proteins sequenced (McPherron and Lee, Proc. Natl. Acad. Sci. USA (1997) 94:12457-12461). The myostatin protein sequence is highly conserved across all of these species. Similarly, the nucleotide sequence for myostatin from mouse, rat, human, baboon, cattle, pig, sheep, chicken and turkey has been determined. See, e.g., U.S. Pat. No. 5,827,733 for the nucleotide sequences of murine and human myostatin; International Publication No. WO 99/02667 for the nucleotide sequence of bovine myostatin; International Publication No. WO 98/33887, for the nucleotide sequences of rat, human, baboon, bovine, porcine, ovine, chicken and turkey myostatin.
The nucleotide sequence of the myostatin gene predicts a protein of about 376 amino acids with a molecular weight of approximately 43 kDa. This protein contains a secretion leader sequence and a proteolytic processing site which releases a 13 kDa peptide, containing 9 cysteine residues. Cloned myostatin expressed in Chinese hamster ovary cells yields two proteins. The first has an apparent molecular weight of about 52 kDa and the second about 15 kDa. Under nonreducing conditions, these proteins appear to be dimers with molecular weights of about 101 kDa and 25 kDa (McPherron et al., Nature (1997) 387:83-90).
Researchers have proposed delivery of mutated myostatin genes to animal subjects for the production of transgenic species having increased muscle tissue. See, e.g., International Publication No. WO 98/33887. However, such approaches pose several drawbacks. For example, because the myostatin gene becomes active during the embryonic stage, reduced myostatin production causes excessive muscle development in utero. Thus, transgenic animals which include mutated genes would likely require cesarean delivery, a serious burden to large animal producers. Additionally, public opposition to genetically engineered animals for human consumption exists and other methods of producing such animals would be desirable.
The present invention is directed to immunological compositions and methods for modulating endogenous myostatin activity in a vertebrate subject. The invention is also useful for treating a number of conditions in vertebrates, including humans and other animals, such as a variety of disorders that cause degeneration or wasting of muscle. Due to the ubiquitous nature of myostatin, the compositions and methods described herein find use in a wide variety of vertebrate subjects, as described further below.
Surprisingly, the invention achieves these results by immunological techniques. It is readily known in the art that immunization against endogenous molecules, such as myostatin, is problematic because the immune system does not recognize such xe2x80x9cselfxe2x80x9d molecules. Thus, the present invention provides a solution to a problem which would normally be encountered when immunizing against an endogenous substance.
Accordingly, in one embodiment, the invention is directed to a myostatin peptide consisting of about 3 to about 100 amino acids. The peptide comprises at least one epitope of myostatin. In preferred embodiments, the myostatin peptide is derived from the region of myostatin spanning amino acids 45 through 376, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36) or amino acids 235 through 376, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36).
In other embodiments, the myostatin peptide has at least about 75% amino acid identity to a peptide comprising an amino acid sequence selected from the group consisting of amino acids 3-18, inclusive of SEQ ID NO:4; amino acids 3-15, inclusive of SEQ ID NO:6; amino acids 3-17, inclusive, of SEQ ID NO:8; amino acids 3-16, inclusive of SEQ ID NO:10; amino acids 3-22, inclusive of SEQ ID NO:12; amino acids 3-25, inclusive of SEQ ID NO:14; amino acids 3-22, inclusive of SEQ ID NO:16; amino acids 3-18, inclusive of SEQ ID NO:20; and amino acids 3-18, inclusive, of SEQ ID NO:22.
In still further embodiments, the invention is directed to a myostatin peptide consisting of about 3 to about 200 amino acids. The peptide comprises at least one epitope of myostatin and is derived from a region of myostatin selected from the group consisting of the region of myostatin spanning amino acids 1 through 350, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); the region of myostatin spanning amino acids 1 through 275, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); the region of myostatin spanning amino acids 25 through 300, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); the region of myostatin spanning amino acids 50 through 325, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); and the region of myostatin spanning amino acids 75 through 350, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36).
In yet further embodiments, the myostatin peptide comprises the amino acid sequence Lys-Arg-Ser-Arg-Arg-Asp (SEQ ID NO:37), the amino acid sequence Lys-Glu-Asn-Val-Glu-Lys-Glu (SEQ ID NO:38) or the amino acid sequence Ser-Leu-Lys-Asp-Asp-Asp (SEQ ID NO:39).
In yet another embodiment, the invention is directed to a myostatin multimer comprising two or more selected myostatin immunogens, wherein each of the immunogens independently comprises at least 3 amino acids defining at least one epitope of myostatin. In particularly preferred embodiments, each of the selected myostatin immunogens comprises at least one epitope of myostatin and independently consists of about 3 to about 200 amino acids, or about 3 to about 100 amino acids, or about 3 to about 30 amino acids, or about 3 to about 15 amino acids.
In other embodiments, each of the selected myostatin immunogens in the multimer independently comprise a selected myostatin peptide as described above. In particularly preferred embodiments, the multimer comprises a molecule with repeating units according to the general formula (MP-X-MP)y, wherein MP is a myostatin peptide, X is selected from the group consisting of a peptide linkage, an amino acid spacer group, a leukotoxin polypeptide and [MP]n, where n is greater than or equal to 1, and y is greater than or equal to 1.
In another embodiment, the invention is directed to a myostatin immunoconjugate comprising at least one myostatin peptide or multimer, as described above, linked to an immunological carrier.
In still further embodiments, the invention is directed to vaccine compositions comprising the myostatin peptide, the myostatin multimer and/or the myostatin immunoconjugate, and a pharmaceutically acceptable excipient.
In yet other embodiments, the invention is directed to polynucleotides encoding the myostatin peptides., the myostatin multimers and the myostatin immunoconjugates above, as well as recombinant vectors comprising the polynucleotides, host cells transformed with the recombinant vectors, and methods of recombinantly producing the myostatin peptides, myostatin multimers and myostatin immunoconjugates.
In other embodiments, the invention is directed to methods of eliciting an immune response against a myostatin immunogen in a vertebrate subject comprising administering the vaccine compositions or polynucleotides above to the vertebrate subject. In particularly preferred embodiments, the immune response elicited reduces endogenous myostatin activity in the vertebrate subject and results in at least one of the following biological effects:
(a) an increase in body weight;
(b) an increase in muscle mass;
(c) an increase in the number of muscle cells;
(d) an increase in the size of muscle cells;
(e) a reduction in body fat content;
(f) an increase in muscle strength;
(g) an increase in mammary gland tissue;
(h) an increase in lactation;
(i) an increase in appetite or feed uptake; or
(j) an increase in the life span of the vertebrate subject.
In other embodiments, the invention is directed to methods of treating a disorder which comprises degeneration or wasting of muscle in a vertebrate subject, the method comprising administering the vaccine compositions or polynucleotides above to the subject. The invention is also directed to methods of modulating GDF11 activity in a vertebrate subject comprising administering the vaccine compositions above.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.